2.5 In vitro wounding assay
A previously described wound healing scratch assay was used to assess cell migration (Das et al. , 2018). Cells were plated at a density of 160000 cells/ml in 24-well culture plates and incubated at 37 °C in 5% CO2 until formation of a confluent monolayer. Cells were then scratched once per well, vertically, with a 200 μL plastic pipette tip to create an artificial wound. After washing twice to remove cellular debris by PBS, cells were treated with 10 ng·mL-1 FGF2, PEG-FGF2 conjugates, or PBS control. The same fields were photographed every 12 h by Nikon Eclipse Ni light microscopy and the areas of cell migration were calculated with ImageJ Software (National Institutes of Health, Bethesda, MD, USA). The percent migration at each time point was defined as the ratio of (the original area - the open area at 24 h) to the original area. All the experiments were performed in triplicate.